Rearrangement of the BCL-2 Gene in Follicular Lymphoma

Efficient DNA extraction from 25-year-old paraffin-embedded tissues: study of 365 samples

“…9,11,14,15,23,24,[30][31][32][33][34] Many studies show that fragmented DNA is obtained due to fixation and embedding processes, only allowing PCR analysis on short target sequences, rarely exceeding 300 bp. 1,3,15,35 Besides characteristics of PETs storage (e.g., duration, temperature, humidity), the most critical step affecting DNA integrity is the fixation process. Specifically, the extent of damage in DNA may depend on the type of fixative used and on the duration of fixation: Bouin's fixative, unbuffered formalin and long fixation time increase DNA degradation.…”

“…A high cycle number was used for PCR amplification (45 cycles), as many authors suggested using 40-50 cycles for amplification of DNA from PETs. 1,11,12,[23][24][25][26] The hot-start PCR conditions were as follows: 10 min at 95uC, followed by 45 cycles of denaturation for 1 min at 95uC, annealing at the appropriate temperature for 1 min (see Table 1) and extension for 30s at 72uC; final extension of 5 min at 72uC. Amplicons were analysed on 2% agarose gels stained with ethidium bromide and visualised by ultraviolet transillumination.…”

“…During formalin fixation, proteinprotein and protein-DNA cross-links occur, and formaldehyde within the tissue gradually changes to formic acid, hydrolysing the DNA. [1][2][3] Similarly, Bouin's fixative, containing picric acid, formalin and acetic acid, damages the integrity of DNA. 3,4 It has been shown that DNA extracted from formalin-fixed PETs can be suitable for polymerase chain reaction (PCR) analysis, as long as high molecular weight DNA is not required.…”

A Method for Assessing Strand Breaks in DNA

“…Many biochemical approaches have also been used to assess chemical damage, such as pyrimidine dimers, N-methylpurine, and cross-linking of DNA (13,14). Damage can also be assessed by the polymerase chain reaction (PCR), using end points ranging from crude assessment of whether the DNA yields any amplified product to precise quantification of the proportion of PCR targets in the sample that are amplifiable (8,15). Precise quantification of damage may take as long as quantification of the target of interest.…”